Since many diseases primarily affect the blood, or blood system, and since many other disorders result in alterations in the blood, hematological analysis, in conjunction with traditional diagnostic techniques, provides a useful clinical tool for the recognition and treatment of many diseases. The automation of this analysis has made its use widespread and commonplace. The compositions and methods of this invention are useful in automated hematological analyses.
In healthy man the circulating blood contains three major categories of cells: a population of small thrombocytes (or platelets) typically around 10 fl in average volume and around 150,000 to 400,000/ul in numerical concentration, the hemoglobin-carrying population of erythrocytes (or red cells) typically around 90 fl in average volume and around 4,000,000 to 6,000,000/ul in numerical concentration, and the population of larger nucleated leukocytes (or white cells). The leukocyte category consists of two numerically predominant or frequent subpopulations (the smaller monomorphonuclear lymphocytes and the larger polymorphonuclear neutrophilic granulocytes--each typically in the range of 1,000 to 5,000/ul in numerical concentration) and three infrequent subpoplulations (the monomorphonuclear monocytes, the polymorphonuclear eisinophilic granulocytes, and the polymorphonuclear basophilic granulocytes each typically below 500/ul in numerical concentration).
In many diseases the three infrequent leukocyte subpopulations tend to be more plentiful. Additionally, in disease, several other even less frequent or anomalous leukocyte subpopulations may appear in the blood in noticeable concentrations. The compositions and methods of this invention permit the enumeration of the two frequent leukocyte subpopulations together with a powerful quantitative screening evaluation of the remaining infrequent physiologic and rare pathologic leukocyte subpopulations.
Electronic cell counters are used in the enumeration of all these blood cell categories.
This enumeration and screening evaluation is achieved by lysing away all the extremely numerous and therefore obscuring erythrocytes (as is usual for resistivity leukocyte counting) and by simultaneously modifying the frequent (and also obscuring) lymphocyte and neutrophil subpopulations so that in terms of relative resistivity size, the lymphocytes are moved further below the infrequent subpopulation cells and the neutrophils are moved further above the infrequent subpopulation cells than are the native unmodified leukocytes of the circulating blood. In this way the two frequent leukocyte subpopulations are drawn apart (as shown in FIG. 1) to expose a clear stage on which pathophysiologic increases of any of the infrequent and rare subpopulations are readily apparent and can be evaluated quantitatively.
From 1960 onwards, it was shown that the use of the proper red cell lysing agent could result in distinguishing white cell subpopulations. See, e.g., Allen, J. D., and Gudaitis, A. V., "Diluting Fluid for Electronic Counting of Leukocytes and Hemoglobin Determinations", Am. J. Clin. Path., 33, 553-556 (1960); Van Dilla, M. A., Fulwyler, M. J. and Boone, I. U., P.S.E.B.M., 125, 367-370 (1967). In these early experiments saponin (sapogenin glycosides) was used as the red cell lysing agent. Unfortunately, saponin required forty-five seconds or more for effective hemolysis. Measurements taken prior to this forty-five second incubation period were inaccurate as a result of red cell stroma interference. Stronger concentrations of saponin would result in only one white cell category, since only the nuclei of all the subpopulations were left. These nuclei have approximately the same size and DNA content thereby inhibiting volumetric or resistivity separation of the white cells into distinct subpopulations.
Another lysing agent, cetrimide (a mixture of quaternary ammonium salts) was tested by prior investigators, resulting in a rough differentiation of white cell subpopulations. See, Hatch, A. and Balazs, T., Am. J. Clin. Path., 36, 220-223 (1960). Further work with cetrimide as a lysing agent illustrated that the choice of the blood diluent played an important role in the results obtained. See D'Angelo, G. and LaCombe, M., "A Practical Diluent for Electronic White Cell Counts", Am. J. Clin. Path., 34, 220-223 (1961). As expected there was interaction not only between the various kinds of blood cells and the lysing reagent, but also between the blood cells and the diluent, the lysing reagent and the diluent, and even the blood proteins and the other three components of the suspension--blood cells, diluent and lysing reagent.
Accordingly, when a further reagent was introduced into certain blood cell analyzers--specifically when, in the 1970's, detergents were introduced into the major diluent or into a wash or blanking line of, for example, those instruments which detected an advancing meniscus level for the purpose of metering the volume of sample suspension which had flowed through the sensor during the analytic cycle--it could be anticipated that there would also be interactions between any introduced detergent and all other components of the diluted blood suspension i.e., blood components (blood cells, blood proteins, blood chemicals and even anticoagulants) and reagents (diluent components, erythrocyte and leukocyte lysate ingredients, hemoglobinometry reagents, and detergent solutions).
This interaction between the environment of blood cells and their ability to maintain their size is well-known. Erythrocytes, because of their lack of granular and nuclear material, are very quickly and dramatically affected by environmental changes; however, it is known that leukocytes and thrombocytes also exhibit this physical environmental response but to a lesser degree.
While the situation is very complex, the main factors that control cell size maintenance in solution are osmolality, pH, conductivity, buffering, ionic size, ionic type, deformation forces, and temperature. Changing one of these parameters can generally be counterbalanced by changing others as well, thus maintaining a solution which keeps the cell volume unchanged. If only one parameter is altered cell volume will usually be affected.
U.S. Pat. Nos. 4,346,018 (filed June 16, 1980), 4,521,518 (filed July 6, 1982) and 4,485,175 (filed Jan. 3, 1983) disclose the use of diluents and lysing agents in the differential volume determination of leukocyte subpopulations. In U.S. Pat. No. 4,485,175, three leukocyte subpopulations (lymphocytes, monocytes, and granulocytes) are differentiated by using a diluent comprising an aqueous solution of procaine hydrochloride, ADA buffer and polynoxylin (dimethylol urea), a distillate of formaldehyde and urea. The lysing agent is described as an aqueous solution of a mixture of quaternary ammonium salts, preferably dodecyltrimethyl ammonium chloride and tetradecyltrimethyl ammonium bromide, and potassium cyanide. The Coulter Counter.RTM. Model S Plus is used to obtain volumetric differentiation of the monocyte and granulocyte subpopulations (combination of neutrophils, basophils, and eosinophils) of leukocytes through the significantly slow addition of the lysing agent, in which the quaternary ammonium salts are present in significantly weak concentrations.
The reagents of the instant invention permit quick and accurate cell volume differentiation, resulting in improved exposure of any or all of the infrequent physiologic and rare pathologic white cell subpopulations. The established cetrimide and derivative lytic reagents and methods require mixtures of quaternary ammonium salts to eliminate red cell fragments (and other debris which result from using the lysing agent) for the separation of monocytes from the three (combined) granulocyte subpopulations. By contrast, the lysing solution of the instant invention contains a single quaternary ammonium salt; this is designed to expose infrequent and rare leukocyte subpopulations. This exposure is obtained by carefully controlling the lytic reaction. Under the conditions disclosed herein, better enumeration of the frequent leukocyte subpopulations as well as exposure of the infrequent and rare leukocyte subpopulations is obtained.
Previously known diluents, when used in conjunction with previously known lysing agents, do not unmask the infrequent and rare leukocyte subpopulations adequately. When these reagents are used the frequent lymphocytes and neutrophils tend to obscure the infrequent and rare subpopulations--an overlap inherent to the reagents. Furthermore, the limited monocyte exposure obtained in the prior art is stable only for a very short period of time. The diluent of the instant invention for the first time utilizes 1,3-dimethylurea (a compound very different from the dimethylolurea of U.S. Pat. No. 4,485,175) as a stabilizing agent which allows all the subpopulations of cells to attain a stable condition in a short period of time and to maintain the stability for long periods relative to the analytical cycle time.
Therefore, it is an object of this invention to provide reagents and methods which rapidly arrive at stable resistivity sizes of white cell subpopulations by carefully controlling the lytic reaction, while providing for adequate lysis of the interfering red cells, and their stroma or ghosts.
It is another object of this invention to provide effective reagents and reliable methods which facilitate the enumeration of the frequent lymphocytes and neutrophils while exposing the infrequent and rare leukocyte subpopulations.
It is a further object of this invention to permit accurate hemoglobinometry fast enough for measurement on an automated system simultaneously with white cell measurement.
It is still a further object of this invention to provide reagents and methods which maintain solution conductivity levels which permit good signal-to-noise ratios for blood-cell counting in automated analytical systems.
It is still another object of the instant invention to provide reaction conditions which permit the erythrocytes and thrombocytes to maintain their volume.